Fragile X syndrome (FXS) is the leading heritable form of mental retardation, and is a leading single-gene disorder associated with autism. FXS is a trinucleotide repeat expansion disorder that generally arises when the CGG element of the fragile X mental retardation 1 (FMR1) gene expands beyond ~200 repeats (full mutation);such expansions are usually accompanied by methylation-coupled silencing. Absence of the FMR1 protein (FMRP) leads to FXS. By contrast, carriers of smaller (permutation;55-200 CGG repeats) alleles have elevated FMR1 mRNA levels in peripheral blood leucocytes and brain, by as much as five- to ten-fold for males with permutation alleles that exceed 100 repeats. We have recently discovered a new disorder, fragile X-associated tremor/ataxia syndrome (FXTAS) that specifically involves premutation carriers;evidence to date suggests that this disorder is caused by a pathologic "gain-of-function" of the elevated FMR1 mRNA itself. More recent evidence suggests that the elevated mRNA may also have neurodevelopment consequences for some children with premutation alleles. The principal objective of the proposed research is to identify the mechanism(s) by which transcriptional activity is increased for alleles in the premutation range, and decreased for alleles in the full mutation range. The motivation for these studies is the eventual correction of the dysregulation in both CGG repeat ranges. We propose to identify the basis for increased transcription for expanded alleles in the premutation range (Aims 1 and 2), and to characterize the properties of the transcriptionally silenced gene for alleles in the full mutation range (Aim 3). These studies will utilize a combination of cloning/transfection strategies, and will capitalize on the vast resource of hundreds of cell pools from patients with alleles spanning normal, premutation, and full mutation ranges. This resource includes dozens of cell pools from methylation and size mosaics, and more than fifty transformed lines, to date, from individuals in all three repeat expansion ranges. The proposed studies should facilitate the development of treatments for fragile X syndrome and for clinical conditions among (premutation) carriers of the fragile X gene. The public health impact of this effort is underscored by the fact that there are estimated to be at least 500,000 premutation carriers, and nearly 100,000 individuals with full mutation forms of the fragile X gene in the US.